Characterization of host-parasite interactions between the oral mucosa and the protozoan Entamoeba gingivalis that drive tissue invasion, destruction and microbial dysbiosis
Final Report Abstract
Periodontitis is an inflammatory disease influenced by oral microorganisms, genetic factors and lifestyle. Entamoeba gingivalis (E.g.), a protozoan commonly found in gingival pockets, may play an important role in the pathogenesis. The aim of this work was to investigate the molecular interactions of E.g. with gingival cell lines and to sequence the genome of the amoeba. Experiments with VAMP3 and VAMP8 knockout cell lines showed that mucin secretion in gingival cells is mediated by VAMP8 and specifically induced by E.g., but, for comparison, not by the oral bacterium Porphyromonas gingivalis (P.g.). We also showed that in gingival fibroblasts the secretion of matrix metalloproteinases (MMPs) is mediated by VAMP3 and both the secretion and activation of MMPs is induced by E.g. infections, but not by P.g. . In gingival cells, E.g. triggers a strong inflammatory response, characterized by IL-8 secretion, which is also mediated by VAMP8. Cytotoxicity tests confirmed the specific pathogenic effect of E.g. on the gingival barrier. In VAMP8 knockout epithelial cells, lacking mucin secretion, the cell death rate after E.g. infection was significantly increased, while infections with P.g. or E.g.-infected connective tissue cells did not show a significantly increased cell death rate. Transcriptome analyses also showed that E.g. infections in gingival epithelial cells strongly increase the expression of inflammation-mediating chemokines such as TNFA and IL-8, while in connective tissue forming fibroblasts the activity of genes that promote cell division is strongly reduced. Electron microscopy showed that E.g. specifically kills living epithelial cells by trogocytosis, whereas this behavior was not observed in fibroblasts. This illustrates the greater pathogenicity of E.g. compared to P.g., as well as cell type-specific, defined pathogenic effects. Contrary to the previous assumption that E.g. does not form cysts, we were able to demonstrate the formation of survival stages that could be induced by in vitro treatment with the antibiotics amoxicillin or metronidazole. The cysts, smaller than trophozoites, had a smooth surface and a chitinous, polygonal wall. This cyst formation could facilitate the persistence and infection of E.g. and make it resistant to periodontal antibiotic treatment. Isolation of pure E.g. DNA from microbially mixed subgingival plaque was achieved by cell sorting using flow cytometry. Sequencing yielded a large dataset of non-bacterial and nonhuman DNA, which is being analyzed for genome assembly. Bacteria that were most commonly phagocytosed by E.g. were P.g. and Treponema spec. This work demonstrates the central role of mucins and VAMP proteins in the defense against E.g. as well as the pathogenetic potential of the parasite through activation of host pro-teases and immune responses and cell type-specific cytotoxicity. The knowledge gained offers approaches for new therapies against periodontitis.
Publications
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Entamoeba gingivalis Exerts Severe Pathogenic Effects on the Oral Mucosa. Journal of Dental Research, 100(7), 771-776.
Bao, X.; Weiner, J.; Meckes, O.; Dommisch, H. & Schaefer, A.S.
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Host prediction for disease-associated gastrointestinal cressdnaviruses. Virus Evolution, 8(2).
Kinsella, Cormac M.; Deijs, Martin; Becker, Christin; Broekhuizen, Patricia; van Gool, Tom; Bart, Aldert; Schaefer, Arne S. & van der Hoek, Lia
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In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment. Frontiers in Cellular and Infection Microbiology, 13.
Becker, Christin; Adam, Aysegül; Dommisch, Henrik; Stach, Thomas & Schaefer, Arne S.
